The progressive salinization of agricultural soils poses a major limitation for the growth and productivity of agricultural crops. Although engineering technology involving drainage and supply of high quality water has been developed to overcome this problem, the existing methods are extremely costly and time-consuming. In many instances, due to the increased need for extensive agriculture, neither improved irrigation efficiency nor the installation of drainage systems is applicable. Moreover, in the arid and semi-arid regions of the world water evaporation exceeds precipitation. These soils are inherently high in salt and require vast amounts of irrigation to become productive. Since irrigation water contains dissolved salts and minerals, application of water further compounds the salinity problem.
Current attempts to enhance the salinity tolerance of model and crop plants are based on conventional breeding and selection of resistant variants. However, such breeding techniques typically require years to develop, are labor intensive and expensive. Moreover, thus far, these breeding and selecting strategies did not result in the mass production of tolerant varieties, suggesting that conventional breeding practices are not sufficient.
An alternative and attractive approach involves the genetic engineering of transgenic crops having enhanced salt tolerance. In recent years, advances in molecular biology have allowed mankind to manipulate the genetic complement of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of the genetic material into plants. Such technology has led to the development of plants with increased pest resistance, plants that are capable of expressing pharmaceuticals and other chemicals and plants that express beneficial agricultural traits. Advantageously, such plants not only contain genes of interest, but remain fertile.
Sodium ions in saline soils are toxic to plants due to their adverse effect on potassium nutrition, cytosolic enzyme activities, photosynthesis and metabolism. Different mechanisms function cooperatively to prevent accumulation of sodium ions (Na+) in the cytoplasm of plant cells, namely restriction of Na+ influx, active Na+ efflux and compartmentalization of Na+ in the vacuole. There is a wide spectrum of plant responses to salinity that are defined by a range of adaptations at the cellular and the whole plant levels, however, the mechanism of sodium transport appears to be fundamentally similar in many plant species. At the cellular level, sodium ions are extruded by plasma membrane Na+/H+ antiporters that are energized by the proton gradient generated by the plasma membrane H+-ATPases (PM H+-ATPases). Cytoplasmic Na+ may also be compartmentalized by vacuolar Na+/H+ antiporters. These transporters are energized by the proton gradient generated by the vacuolar H+-ATPase and H+-PPiase.
The response of plants to salt stress has previously been studied in model plant species with sequenced genomes, including Arabidopsis thaliana and in rice (Goff S A, et al. (2002) Science 296:92-100; Yu J, et al. (2002) Science 296:79-92). Differential genomic screens carried out in Arabidopsis and rice have shown that plants respond to salt stress by up-regulation of a large number of genes involved in diverse physiological functions.
PM H+-ATPases are the primary ion pumps in plasma membranes of plants and fungi. They are encoded by a large multigene family, amounting to 12 members in the salt-sensitive plant Arabidopsis thaliana alone. PM H+-ATPase isoforms are expressed in different tissues and control diverse physiological functions (Palmgren M G (2001) Ann Rev Plant Physiol Plant Mol Biol 52:817-45; Sekler I & Pick U (1993) Plant Physiol 101:1055-1061). In yeast such as Saccharomices pombe, PM H+-ATPases energize Na+ extrusion via a Na+/H+ antiporter by generating the protomotive force across the plasma membrane. A large body of evidence suggests that PM H+-ATPases also contribute to salinity tolerance. In plants, salt stress induces activation and enhanced expression of PM H+-ATPases, either by over-expression of specific enzyme isoforms, or by activation of existing enzymes (Reuveni M, Bressan R A & Hasegawa P M 1993 J Plant Physiol 142:312-318); Zhang J S et al., (1999) Theor Apll Genet. 99:1006-1011; Kerkeb L, Donaire J P & Rodriguez-Rosales M P (2001) Physiologia Plantarum 111:483-490). Two specific isoforms of PM H+-ATPases encoding genes were identified in tomato and in A. thaliana which are specifically involved in the response to salt stress (Kalampanayil B D and Wimmers L E (2001) Plant, Cell, Environment 24:999-1005; Vitart V et al., (2001) The Plant J 27:191-2001).
A comparison of ion distribution in cells and tissues of various plant species indicates that a primary characteristic of salt-tolerant plants is their ability to exclude sodium out of the cell and to take up sodium and sequester it in the cell vacuoles (Niu, X., et al., 1995 Plant Physiol. 109:735-742). This strongly suggests that Na+/H+ antiporter from salt-tolerant plants have functionally more effective sodium transport systems compared with salt-sensitive plants such as Arabidopsis. 
Several sodium transport systems associated with salt tolerance have been characterized in different organisms and a few of the genes involved in this process have been identified and used to generate plants having enhanced salt-resistance. For example, a homologue of sodium antiporter (AtNhx1) from the salt-sensitive plant Arabidopsis thaliana has been identified and characterized. Over expression of AtNhx1 in Arabidopsis (Apse, M P et al., (1999) Science 285:1256-1258) as well as in fission yeast shows increased salt tolerance due to better performance of salt compartmentation into the vacuole. Zhang et al have shown that over expression of vacuolar Na+/H+ antiporter in A. thaliana and tomato plants led to a significant enhancement in salinity tolerance (Zhang H X & Blumwald E (2001) Nature Biotechnology 19:765-768). Shi et al demonstrated that over expression of Na+/H+ antiporter SOS1 in plant plasma membranes improves salinity tolerance in A. thaliana, suggesting that a plasma membrane-type Na+/H+ antiporter is essential for plant salt tolerance. (Shi H, Lee B H & Zhu J K (2003) Nat Biotechnology 21:81-85).
International Patent Application No. WO 91/06651 discloses a single gene (sod2) encoding for a Na+/H+ antiporter that has been shown to confer sodium tolerance in fission yeast, although the role of this plasma membrane-bound protein appears to be only limited to yeast.
US Patent Application No. 20040040054 discloses polynucleotides encoding plant Na+/H+ antiporter polypeptides isolated from Physcomitrella patens and methods of applying these plant polypeptides to the identification, prevention, and/or conferment of resistance to various plant diseases and/or disorders, particularly environmental stress tolerance in plants, specifically salt stress.
US Patent Application No. 2002178464 discloses transgenic plants transformed with exogenous nucleic acid which alters expression of vacuolar pyrophosphatases in the plant, wherein the transgenic plants are tolerant to a salt. Specifically, the exogenous nucleotide encodes a vacuolar pyrophosphatase H+ pump, AVP1.
International Patent Application No. WO 03/031631 discloses nucleic acids and nucleic acid fragments encoding amino acid sequences for salt stress-inducible proteins, protein phosphatases mediating salt adaptation in plants, plasma membrane sodium/proton antiporters, salt-associated proteins, glutathione peroxidase homologs associated with response to saline stress in plants, and early salt-responding enzymes such as glucose 6-phosphate 1 dehydrogenase and fructose-biphosphate aldolase in plants and the use thereof for, inter alia, modification of plant tolerance to environmental stresses and osmotic stresses such as salt stress modification of plant capacity to survive salt shocks, modification of compartmentalization of sodium in plants, for example into the plant cell vacuole, modification of sodium ion influx and/or efflux, modification of plant recovery after exposure to salt stress, and modification of plant metabolism under salt stress.
These studies demonstrate that, using combination of breeding strategies and genetic manipulation, it is be possible to generate plant crop having enhanced salt tolerance. However, all of the aforementioned methods rely on the isolation, characterization and over expression of genes from plant sources, and accordingly the success of such approaches relies on the intrinsic adaptation of the plant genetic material, and the encoded proteins, to salt environment. Since plants are not well adapted to highly saline conditions, the success of these approaches has been limited.
Exceptionally salt tolerant (halotolerant) organisms may provide useful for identification of basic mechanisms that enhance salinity tolerance. A special example of adaptation to variable saline conditions is the unicellular green algae Dunaliella, a dominant organism in many saline environments, which can adapt to practically the entire range of salinities. Dunaliella responds to salt stress by massive accumulation of glycerol (its internal osmotic element), enhanced elimination of Na+ ions, and accumulation of distinct proteins (Pick U et al. In A Lauchli, U Luthge, Eds. Salinity: Environment-Plants Molecules, Ed Acad. Pub. Dordrecht. Kluwer, pp 97-112, 2002). Since the cells of this genus do not possess a rigid cell wall, they respond to changes in salt concentration by rapid alterations in cell volume and then return to their original volume as a result of adjustments in the amounts of intracellular ions and glycerol. It has been reported that the adaptation to extreme salinity involves short-term and long-term responses. The former include osmotic adjustment by accumulation of large amounts of intracellular glycerol and efficient elimination of Na+ ions by plasma membrane transporters. The latter involves synthesis of two extrinsic plasma membrane proteins, a carbonic anhydrase and a transferrin-like protein. These proteins are associated with acquisition of CO2 and Fe, respectively, whose availability is diminished by high salinity. In addition, Ajalov et al reported on the isolation of a 64 kDa and 28 kDa salt-induced polypeptides from Dunaliella salina (Ajalov et al. (1996), Biochemical Society Transactions, 24(4), 5345).
Due to its remarkable ability to adapt to highly saline conditions, Dunaliella serves as a valuable model for the identification of basic mechanisms in salinity tolerance.
The success of current plant breeding strategies which are based on genetic manipulation of genes from plant sources has been limited due to the limited capability of plants to adapt to saline conditions. There remains a need in the art to develop genetic engineering approaches that are superior to current techniques, and that would yield transgenic plants having high salt tolerance that are capable of growing in conditions of high salinity.